1) Sensory nerve-derived vascular branching patterning signals in the limb skin We previously observed that arterial vessels and peripheral sensory nerves (PNs) develop alongside each other in the embryonic limb skin. This co-patterning is developed by PN-mediated signal(s) that instructively guide the arterial branching network (Mukouyama et al. 2002). Therefore, the limb skin vasculature affords an attractive system in which to study the nature of neuronal signals that control vascular network formation. Using the tissue-specific knockout technology, we have begun to dissect out the PN-derived signals that participate in integrating both branching networks. We showed that PN-derived vascular endothelial growth factor (VEGF)-A functions to control arteriogenesis-arterial differentiation and smooth muscle cell association (Mukouyama et al. 2005). We have recently discovered that PN-derived C-X-C motif chemokine ligand (CXCL) 12 controls the nerve-blood vessel alignment (manuscript in preparation). Taken together, the limb skin vasculature model allows us to unravel mechanisms that coordinate arterial differentiation and patterning of vascular branching. However, what controls venous differentiation and patterning of venous and lymphatic vessel branching still remains to be answered. We are engaged in a new project for studying the role of the neuro-vascular association during tissue repair or in disease conditions. Whole-mount immunofluorescence microscopy has revealed that adult ear skin maintains the neuro-vascular bundle, suggesting that the association reflects the mutual requirement of nerve and vessel in the function and maintenance of both networks. Using this adult ear skin vasculature model, we are currently studying peripheral nerve regeneration and re-vascularization in the ear skin regeneration/wound healing. 2) Lymphatic vessel development in the central nervous system Central Nervous System (CNS) has been identified as an immunoprivileged site originally due to the lack of lymphatic vasculature. There are intricate vascular network without lymphatic vasculature in the brain and spinal cord during development and in adult. Using the spinal cord vasculature model, we focus on understanding mechanisms by which the spinal cord prevents lymphangiogenesis but not angiogenesis. We have found that supernatant from cultured embryonic spinal cord prevents the differentiation of lymphatic endothelial cells, as well as the migration of lymphatic but not blood endothelial cells. Now we are extensively working to identify neuronal or glial signal(s) that prevent the differentiation and migration of the lymphatic endothelial cells in the CNS. The findings also provide a rational for testing the signal(s) as potent inhibitors of adult and tumor-induced lymphangiogenesis. 3) Interaction of developing coronary vasculature with cardiac sympathetic neurons A favorable new system for studying the neuro-vascular association has been identified that in the embryonic heart, coronary vasculature has sympathetic innervations to control vasodilatation or constriction. We have recently found that coronary vascular remodeling and patterning precedes cardiac innervations and the coronary patterning helps to guide axonal projection (manuscript in preparation). Cardiac sympathetic axons fail to outgrow in the mutants having defective coronary development, whereas normal coronary development occurs in the absence of cardiac sympathetic axons. Further in vitro studies have demonstrated that coronary smooth muscle cells provide guidance cues for axons. We hope to determine the coronary-derived signal(s) that cause coronary vessels and cardiac sympathetic axons to interact. 4) Vascular niche for adult neurogenesis Stem cells are established in the niche, a unique and specialized microenvironment. Given the importance of the vascular niche for a variety of stem cells, understanding the paracrine signals for stem cell maintenance has become more important. Whole-mount staining approach of the subventricular zone (SVZ) in the lateral ventricle walls of adult brain has revealed that slowly-dividing SVZ cells (adult neural stem cells?) are closely interacted with the local vasculature. Our challenge is to utilize a systematic multi-faceted approach to identify and validate the vascular niche signals involved in maintenance, self-renewal, proliferation and differentiation of neural stem cells. We are now evaluating the physiological relevance of the candidate niche signals in vitro using neurosphere stem cell assay. Furthermore, we plan to examine the SVZ neurogenesis in knockout mice to provide a definitive test of the requirement for a particular candidate niche gene.